An anti-IgE monoclonal antibody that binds to IgE on CD23 but not on high-affinity IgE.Fc receptors
Introduction
Allergic diseases, such as allergic asthma, allergic rhinitis, and food allergy, are very prevalent among populations living a Westernized lifestyle (Gold and Wright, 2005, Isolauri et al., 2004). Numerous clinical studies of the humanized anti-IgE antibody, omalizumab, in many allergic diseases have shown that omalizumab is efficacious and safe in treating patients with those allergic diseases (Lanier et al., 2003, Milgrom et al., 1999, Rafi et al., 2010). These results have not only confirmed that IgE is a central mediator in the pathogenesis of many allergic diseases but have also validated IgE as a good molecular target for therapeutic intervention (Holgate et al. 2005). The widely recognized pharmacologic mechanisms of omalizumab are its ability to neutralize free IgE and to inhibit IgE binding to FcɛRI on basophils and mast cells (Chang, 2000, Chang et al., 2007). Moreover, the depletion of free IgE in the blood and in interstitial spaces causes a down-regulation of FcɛRI on basophils and mast cells over time (Beck et al., 2004, Chang and Shiung, 2006). While the ability of the antibodies to bind to membrane-bound IgE (mIgE) (Davis et al. 1993), which forms part of the B cell receptor complex (Niiro and Clark 2002), and to cause the lysis of IgE-expressing B cells and hence inhibit IgE production, was considered in the original design of the anti-IgE therapeutic antibodies, this property has not been confirmed for omalizumab in patients.
Therapeutic approaches aiming at suppressing IgE production, such as anti-interleukin 4 administration (Hart et al. 2002), TH2/TH1 modulation (Stokes and Casale 2004), and anti-CD23 treatments (Bonnefoy et al., 1990, Poole et al., 2005), have drawn much interest among researchers attempting to develop treatments for IgE-mediated allergic diseases. CD23 expressed on the surface of B cells, which binds to IgE with an association constant Ka of about 108–109 M−1 (Gould and Sutton 2008), is known to be involved in the regulation of IgE production (Conrad et al. 2007). A mature CD23 molecule is formed by three 45-kDa type II transmembrane subunit proteins that belong to the C-type lectin family (Conrad 1990). Each subunit contains an α-helical stalk region between the extracellular lectin domain and the transmembrane domain, which bundles to form coiled-coil trimers (Beavil et al., 1992, Hibbert et al., 2005). Membrane-bound CD23 is cleaved by a metalloproteinase, ADAM10, releasing the secreted form of CD23 (Lemieux et al. 2007). It has been shown in mice in vivo that when the lectin domain of membrane-bound CD23 is bound by IgE or by an anti-CD23 antibody, CD23 becomes more stable and is less susceptible to the cleavage by ADAM 10, which signals the B cell to reduce IgE synthesis (Conrad et al., 2007, Ford et al., 2006). Several chimeric anti-CD23 mAbs were shown to cross-link CD23 and inhibit the transcription of ɛ gene and the production of IgE by human peripheral B cells in culture (Yabuuchi et al. 2002). An anti-CD23 mAb, lumiliximab, was shown to induce apoptosis by cross-linking CD23 on B cell lines or on chronic lymphocytic leukemia cells (Pathan et al. 2008). Furthermore, lumiliximab was able to reduce blood IgE levels in allergic patients (Rosenwasser et al. 2003) and is potentially applicable for treating patients with chronic lymphocytic leukemia (Byrd et al. 2007).
In this study, we have developed an anti-IgE mAb, 8D6, which resembles omalizumab in various aspects, including being able to bind to the same region on the CH3 domain of ɛ chain, not being able to bind to IgE on FcɛRI, and not being able to induce the activation of basophils. However, mAb 8D6, unlike omalizumab, has the additional attribute of being able to bind to IgE that is already bound by CD23 and hence has the potential to down-regulate IgE synthesis.
Section snippets
Cells
Mouse NS0 myeloma cells and hybridoma cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS, 4 mM l-glutamine, 50 μg/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen). The RBL SX-38 cell line, provided by Dr. J.P. Kinet of Harvard Medical School, was maintained in MEM (Invitrogen) supplemented with 10% heat-inactivated FBS, 2 mM GlutaMAX (Invitrogen), 25 mM HEPES, 1 mM sodium pyruvate, 50 μg/ml penicillin, and 100 μg/ml streptomycin.
Generation of anti-human IgE mAbs that bind to IgE on FcɛRII but not FcɛRI
To obtain anti-human IgE mAbs with the desired set of binding specificities as described in the introduction, BALB/c mice were immunized with human mIgE.FcL, and hybridoma clones were prepared. In the primary screening procedure, the clones specific for human IgE in the initial ELISA were tested for their ability to compete with omalizumab for binding to IgE. Those that did compete with omalizumab were then screened for their ability to bind to human IgE-pulsed CD23-expressing SKW cells by
Discussion
The anti-IgE therapeutic antibody, omalizumab, has been approved in the United States since 2003 for the treatment of moderate to severe allergic asthma and in the European Union since 2005 for the treatment of severe allergic asthma (Chang et al. 2007). Many patients have been given the same omalizumab dosage continuously for several years without a clearly defined treatment period (Lanier 2006). The general understanding among the clinicians prescribing omalizumab is that IgE is continuously
Acknowledgements
We thank Dr. Bing-Hung Chen for helping us to obtain experimental materials from Dr. D.H. Conrad. This study was supported by a grant, NSC99-2320-B-001-006-MY3, from the National Science Council, Taiwan.
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